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hHR23B is the human homologue of the yeast protein RAD23 and is known to participate in DNA repair by stabilizing xeroderma pigmentosum group C protein. However, hHR23B and RAD23 also have many important functions related to general proteolysis. hHR23B consists of N-terminal ubiquitin-like (UbL), ubiquitin association 1 (UBA1), xeroderma pigmentosum group C binding, and UBA2 domains. The UBA domains interact with ubiquitin (Ub) and inhibit the assembly of polyubiquitin. On the other hand, the UbL domain interacts with the poly-Ub binding site 2 (PUbS2) domain of the S5a protein, which can carry polyubiquitinated substrates into the proteasome. We calculated the NMR structure of the UbL domain of hHR23B and determined binding surfaces of UbL and Ub to UBA1, UBA2, of hHR23B and PUbS2 of S5a by using chemical shift perturbation. Interestingly, the surfaces of UbL and Ub that bind to UBA1, UBA2, and PUbS2 are similar, consisting of five beta-strands and their connecting loops. This is the first report that an intramolecular interaction between UbL and UBA domains is possible, and this interaction could be important for the control of proteolysis by hHR23B. The binding specificities of UbL and Ub for PUbS1, PUbS2, and general ubiquitin-interacting motifs, which share the LALA motif, were evaluated. The UBA domains bind to the surface of Ub including Lys-48, which is required for multiubiquitin assembly, possibly explaining the observed inhibition of multiubiquitination by hHR23B. The UBA domains bind to UbL through electrostatic interactions supported by hydrophobic interactions and to Ub mainly through hydrophobic interactions supported by electrostatic interactions.

Abstract: The human DNA-dependent activator of IFN-regulatory factor (DAI) protein, which activates the innate immune response in response to DNA, contains two tandem Z-DNA binding domains (Zα and Zβ) at the NH2 terminus. The hZβDAI structure is similar to other Z-DNA binding proteins, although it demonstrates an unusual Z-DNA recognition. We performed NMR experiments on complexes of hZβDAI with DNA duplex, d(CGCGCG)2, at a variety of protein-to-DNA molar ratios. The results suggest that hZβDAI binds to Z-DNA via an active-di B–Z transition mechanism, where two hZβDAI proteins bind to B-DNA to form the hZβDAI–B-DNA complex; the B-DNA is subsequently converted to left-handed Z-DNA. This novel mechanism of DNA binding and B–Z conversion is distinct from Z-DNA binding of the human ADAR1 protein. [Copyright &y& Elsevier]

Abstract: The Yaba-like disease viruses (YLDV) are members of the Yatapoxvirus family and have double-stranded DNA genomes. The E3L protein, which is essential for pathogenesis in the vaccinia virus, consists of two domains: an N-terminal Z-DNA binding domain and a C-terminal RNA binding domain. The crystal structure of the E3L orthologue of YLDV (yabZαE3L) bound to Z-DNA revealed that the overall structure of yabZαE3L and its interaction with Z-DNA are very similar to those of hZαADAR1. Here we have performed NMR hydrogen exchange experiments on the complexes between yabZαE3L and d(CGCGCG)2 with a variety of protein-to-DNA molar ratios. This study revealed that yabZαE3L could efficiently change the B-form helix of the d(CGCGCG)2 to left-handed Z-DNA via the active-mono B–Z transition pathway like hZαADAR1. [ABSTRACT FROM AUTHOR]

Abstract: The Zα domain of human ADAR1 (ZαADAR1) preferentially binds Z-DNA rather than B-DNA with high binding affinity. ZαADAR1 binds to the Z-conformation of both non-CG-repeat DNA duplexes and a d(CGCGCG)2 duplex similarly. We performed NMR experiments on complexes between the ZαADAR1 and non-CG-repeat DNA duplexes, d(CACGTG)2 or d(CGTACG)2, with a variety of protein-DNA molar ratios. Comparison of these results with those from the analysis of d(CGCGCG)2 in the previous study suggests that ZαADAR1 exhibits the sequence preference of d(CGCGCG)2 ≫d(CACGTG)2 >d(CGTACG)2 through multiple sequence discrimination steps during the B–Z transition. [ABSTRACT FROM AUTHOR]